Interacting cascades of signaling proteins, particularly kinases, play crucial roles in the development and maintenance of cancer. Despite their importance in oncogenic signal transduction pathways, little is understood about the biochemical behavior of these proteins within the context of the living cell. It is now clear that these signaling cascades are not merely a linear series of enzymatic reactions leading from discrete stimuli to defined cellular responses. Rather, they are interrelated in complex networks such that their biochemical properties within the living cell are not intuitive. Traditional biochemical studies that rely on lysates of bulk cell populations cannot decipher the inherent properties of cell signaling due to averaging of heterogeneous cellular responses across the population. To understand such biochemical phenomena as cross-talk between pathways, thresholds of activation and bistable states, kinase assays must be performed in intact, single cells. The current work is directed at decoding the biochemical behaviors of the Ras-related pathways- signaling pathways of particular import to cancer biology. These studies will apply a powerful new technology for kinase assays in single cells to a unique series of tumor cell lines possessing constitutively active or inactive signaling proteins. The cell lines (human HT1080/MCH603 fibrosarcoma and mouse NIH3T3) display a range of tumorigenic characteristics dependent on the repertoire of constitutive activity in Ras-related signaling pathways. In mass populations of MCH603 cells our data support the notion that the threshold behavior and crosstalk among these pathways closely relate to the relative aggressiveness of their tumorigenic phenotype. The current studies will reveal how the behavior of signaling cascades involving the kinases phosphoinositide-3-kinase (PI3K), Akt, p21-activated kinase and Erk1/2 interrelate in living, single cells. The specific aims for this project are to 1) determine whether induction of crosstalk, generated by P13K activity, requires a threshold level of activated PI3K; 2) determine if cross-activation of these kinase pathways is coordinate or sequential; and 3) determine if cross-talk activation of MAP kinase cascades is reversible or irreversible after withdrawal of the PI3K or Akt stimulus. These studies will provide additional information relating to our understanding of the consequences of activation of kinase signaling pathways with respect to malignant transformation, aggressive tumor growth and survival.